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Abstract

Background

Scleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal
acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory
and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality
in SSc and an unmet medical challenge, mostly because of our limited understanding
of the molecular factors and signalling events that trigger and sustain disease progression.
Recent evidence has correlated skin fibrosis in SSc with stabilization of proto-oncogene
Ha-Ras secondary to auto-antibody stimulation of reactive oxygen species production.
The goal of the present study was to explore the molecular connection between Ha-Ras
stabilization and collagen I production, the main read-out of fibrogenesis, in a primary
dermal fibroblast culture system that replicates the early stages of disease progression
in SSc.

Results

Forced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the
promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity
of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous
collagen I proteins. Moreover, normal levels of Tgfβ transcripts and active transforming growth factor-beta (TGFβ) implied Ha-Ras stimulation
of the canonical Smad2/3 signalling pathway independently of TGFβ production or activation.
Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation
and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation
and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and
collagen I production in cells that constitutively express Ha-Ras.

Conclusions

Our study shows for the first time that constitutively elevated Ha-Ras protein levels
can directly stimulate Smad2/3 signalling and collagen I accumulation independently
of TGFβ neo-synthesis and activation. This finding therefore implicates the Ha-Ras
pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic
targets in SSc.

Background

Wound healing is a complex and tightly regulated physiological process that involves
several different cell types and a plethora of signalling molecules [1-3]. In the early phase of this process, platelets brought by the blood stream form a
fibrin cloth at the site of injury that blocks bleeding (haemostasis). Increased levels
of soluble signals, induced by the cell-mediated inflammatory response, subsequently
promote migration and proliferation of angiogenic cells and activated fibroblasts
(myofibroblasts) that synthesize extracellular matrix (ECM) proteins, chiefly collagen
I [1]. By contracting the newly synthesized ECM, myofibroblasts allow the closure of the
wound where the provisional matrix is ultimately remodelled to form a scar [1]. Failure of myofibroblasts to terminate the wound healing process results in excessive
accumulation and contraction of a poorly organized ECM. Unopposed myofibroblasts activation
in fibrotic conditions, such as scleroderma (SSc), causes gradual and irreversible
alteration of connective tissue architecture with deleterious consequences for organ
function. In spite of significant investigative effort, our current knowledge of the
molecular and cellular events that promote and sustain myofibroblasts activation is
limited and consequently, the clinical management of affected patients remains confined
to therapies that alleviate secondary symptoms rather than arresting the often fatal
consequences of the fibrotic response.

Clinical findings, cell culture experiments and animal models have firmly established
the prominent role that transforming growth factor-β (TGFβ) plays in modulating the
physiological process of wound healing and in driving the pathological sequence of
fibrotic responses [2,3]. Even though genetic or pharmacological interference of TGFβ signalling in rodents
can mitigate fibrotic disease, they can also result in severe side effects due to
the wide range of biological processes that involve this multifunctional cytokine
[2]. It follows that a better understanding of molecular events upstream, downstream
or parallel to improper TGFβ signalling represents a pre-requisite to the development
of more effective and safer therapies for fibrotic conditions.

TGFβ signals through the activation of a membrane-receptor serine/threonine kinase
complex that phosphorylates the Smad2 and Smad3 proteins [receptor-activated Smads
(R-Smad); canonical TGFβ signalling pathway] [4]. Activated R-Smad proteins associate with Smad4 to migrate into the nucleus and modulate
the expression of several different genes together with transcriptional co-activators
and co-repressors [4]. In addition to the canonical R-Smad pathway, TGFβ can also stimulate the activity
of mitogen-activated protein kinases (MAPKs; non-canonical TGFβ signalling pathway)
and MAPKs and other stress response pathways can, in turn, modulate R-Smad signalling
with discrete intracellular outcomes [5]. For example, the proto-oncogene Ha-Ras can stimulate or inhibit R-Smad signalling,
operate upstream of TGFβ by promoting its auto-induction or act independently of the
canonical TGFβ signalling pathway [6-10]. Hence, complex interactions amongst different signalling pathways are believed to
specify contextual responses of the cells to diverse environmental stimuli.

The Ras gene family comprises three genetically distinct but structurally related
proteins (Ha-Ras, Ki-Ras and N-Ras), which operate as molecular switches that cycle
between an inactive GDP (guanosine diphosphate)-bound to an active GTP (guanosine
triphosphate)-bound form [11]. Ras family members have functionally distinct roles that are dictated by their intracellular
localization and the cellular context [11]. Ras signalling is the nodal point of multiple extracellular cues, including the
profibrotic signals of TGFβ, angiotensin II, platelet-derived growth factor (PDGF)
and reactive oxygen species (ROS) [7,12-14]. Recent studies of SSc cells have causally connected circulating auto-antibodies
against PDGF receptors (PDGFR) with the stimulation of ROS production, Ha-Ras stabilization
and collagen I overproduction [15,16]. However, the contribution of Ha-Ras activity to SSc fibrogenesis, as well as the
cross-talk between Ha-Ras and TGFβ signalling in this disease process remains to be
fully explored.

It was the scope of the present study to investigate the pro-fibrotic potential of
Ha-Ras signalling by using a cell culture system that replicates the downstream events
previously described in SSc myofibroblasts. Our results show for the first time that
constitutively elevated Ha-Ras protein levels promote R-Smad signalling and collagen
I accumulation independently of TGFβ synthesis and activation. These findings implicitly
connect the Ha-Ras pathway with the onset of fibrosis through the stimulation of canonical
TGFβ pathway, even though the biochemical identity of the connection was not investigated
here. More generally, our work provides new insight into early disease-causing events
that could in principle represent new therapeutic targets in fibrotic conditions like
scleroderma.

Materials and methods

Cell cultures

Human fetal dermal fibroblasts (hDF; GM06111) were purchased from the Human Genetic
Mutant Cell Repository (NJ, USA). Cells were maintained at 37°C in a sterile and humidified
atmosphere of 5% CO2. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal
bovine serum (FBS; Atlanta Biologicals, GA, USA) and supplemented with streptomycin,
penicillin and fungizone. Primary mouse dermal fibroblast (mDF) cultures were established
from the dorsal skin of 4-day-old wild-type mice and grown as described above. Several
8 mm sterile skin punches were made from each newborn mice, freed of the subcutaneous
tissue by scraping, and laid flat individually into a 10-cm2 tissue culture plate with the dermal side down. Explants were incubated at 37°C for
5 min to let skin adhere. Ten millilitres of medium was added into each plate and
cells were allowed to migrate out of the explants for 10 days. Once confluent, cells
were trypsinized and either stored in liquid nitrogen or employed immediately: in
both cases, cells between passages 1 and 3 were used.

Cell transfections

Primary mDF and hDF were seeded the day before transfection at a density of 10,000
cells/cm2 and cultured in 0.2% FBS. Cells were transiently co-transfected using Lipofectamine
2000 (Invitrogen, CA, USA) with 0.5 ng of the control plasmid SV40:Renilla-Luc (Promega,
WI, USA) and 200 ng of the COL1A2 (human pro-α2 (I) collagen gene) reporter plasmid
containing wild-type or mutant TbRE (TGFβ-responsive element) sites [17] or the Smad3 responsive plasmid (CAGA)12MLP-Luc (a kind gift of Dr Joan Massagué). In some experiments, the COL1A2 reporter
was transiently co-transfected with plasmids expressing wild-type or constitutively
active (V12 variant) proto-oncogene Ha-Ras (Ha-Ras/pSG5 and ca-Ras/pSG5, respectively) or dominant-negative
(N17 variant) Ha-ras (DN-Ras/pSG5; kindly provided by Dr Enrico Avvedimento), or with
a plasmid expressing dominant-negative (MH2 deletion) Smad3 (DN-Smad3) [17]. In other transfection experiments, hDF cultures were treated with 20 μg/mL neutralizing
pan-TGFβ antibody (MAB1835, R&D System, MN, USA). Luciferase assays were performed
16 h and 24 h after cell transfection and the results were evaluated as previously
described [17]. Statistical analyses were performed for all of the experiments using Student's t test, assuming a P value of ≤ 0.05 as significant.

Lentiviral infections

A lentivirus expressing the wild-type proto-oncogene Ha-Ras was generated by mutating
Ha-Ras V12 coding sequence using the quick change II site direct mutagenesis kit (Stratagene,
CA, USA) following the manufacturer's instructions. Ha-Ras coding sequence was subcloned
into the VVCW/BE lentiviral expression plasmid after EcoR1/EcoRV double digestion.
Ha-Ras/VVCW/BE or VVCW/BE empty vector were cotransfected with CMVδR8.2 and pMD.G
vectors into 293T packaging cell line as described previously [18]. Viral supernatants were collected 48 h and 72 h after transfection and used to infect
mDF cells in the presence of 10 μg/mL of polybrene.

Immunoblots and immunocyotochemistry

mDF cultured for 2 days in 0.2% FBS were infected with Ha-Ras expressing and control
lenti-particles for the indicated length of time. In some experiments, the culture
medium included neutralizing pan-TGFβ antibody or MEK inhibitor (PD98059) at concentrations
of 10 μM (Calbiochem-EMD Biosciences, NJ, USA). Cell layers were scraped into ice-cold
Tris-buffered saline solution (pH 7.4) and flash frozen in liquid nitrogen. Cell extracts
were prepared and assayed for total protein content using the BCA kit (Pierce, IL,
USA). Protein extracts (10-25 μg/lane) were fractioned by 10% or 6% (w/v) SDS-PAGE
and electroblotted onto an Immobilon-P membrane (Millipore, MA, USA). Membranes were
incubated first with antibodies against p-Smad3 (Invitrogen) or Smad3 (Zymed; 1:1000 dilution) and subsequently with HRP-conjugated
anti-rabbit IgG antibody (1:25,000 dilution; Jackson ImmunoResearch Laboratories,
PA, USA). Immunoreactive products were visualized by chemiluminescence using the ECL
Plus kit (Amersham Biosciences, Amersham, UK) and their relative intensity was evaluated
with the aid of Adobe Photoshop software (Adobe Systems Inc, CA, USA). Actin myofibres
were visualized in cells infected with Ha-Ras and control lentivirus using antibodies
against α-smooth muscle actin (αSMA; Chemicon, CA, USA).

Results

The first set of experiments was designed to test the hypothesis that wild-type proto-oncogene
Ha-Ras (hereafter referred solely as Ha-Ras) is directly involved in collagen I stimulation
and to investigate the underlying mechanism. To this end, primary fetal hDFs were
transiently co-transfected with a vector expressing constitutively active Ha-Ras (ca-Ras)
and a luciferase reporter plasmid driven by the COL1A2 proximal promoter. The results
demonstrated that ca-Ras over-expression results in a ~sixfold increase of COL1A2
promoter activity (Figure 1A). Ha-Ras over-expression similarly resulted in increased COL1A2 promoter activity
but to a lesser extent than ca-Ras, perhaps reflecting the inherent activation of
the latter compared to the former protein (Figure 1A). Reduced COL1A2 up-regulation in hDF co-expressing Ha-Ras and dominant-negative
Ha-Ras (DN-Ras) demonstrated the specificity of the former protein action (Figure
1A). Moreover, comparable amounts of the various Ha-Ras versions excluded the formal
possibility that the activity of individual expression constructs might account for
the observed changes in the transcription from the COL1A2 promoter (Figure 1B).

The COL1A2 proximal promoter contains a TGFβ responsive element (TbRE) that mediates
transcriptional up-regulation through the binding of a multiprotein complex that includes
R-Smads, Sp1 and p300/CBP and that is also targeted by other pro-fibrotic stimuli,
such as those trigged by acetaldehyde, sphingolipids and oncostatin M. [3]. Accordingly, we assessed the potential involvement of R-Smad pathway and the TbRE
in Ha-Ras-mediated collagen up-regulation. Two lines of evidence strongly suggested
that Ha-Ras stimulates COL1A2 expression, in part, through the binding of activated
R-Smad complexes to the TbRE. First, mutations in the Smad3-binding site of the TbRE
significantly reduced ca-Ras up-regulation of the COL1A2 promoter (Figure 1C); second, co-transfection of a DN-Smad3 expression plasmid abrogated ca-Ras ability
to increase COL1A2 promoter activity (Figure 1C). Once again, immunoblots documented appreciable levels of recombinant DN-Smad3 (Figure
1D).

The findings of the transient cell transfection experiments were confirmed in quiescent
mDFs that were stably infected with a lentivirus construct expressing Ha-Ras. Specifically,
these analyses showed that Ha-Ras expressing mDF display augmented Smad3 phosphorylation
and Smad3-reporter plasmid activity (Figure 2A) and collagen I production and COL1A2 promoter transcription (Figure 2B and 2F), as well as more cells expressing contraction-competent actin myofibres compared
to mDF infected with control lentivirus (Figure 2C). An additional outcome of Ha-Ras overexpression in mDF cells included the rapid
activation of ERK1/2 signalling (Figure 2A), which is part of the self-propagating loop of ROS production, Ha-Ras stabilization
and collagen I accumulation in SSc [15,16] and also of JNK and p38 MAPK (data not shown). As a result of its participation in
SSc pathogenesis [15,16], ERK1/2 signalling in Ha-Ras over-expressing mDF cells was blunted with the MEK inhibitor
PD98059. However, ERK1/2 inhibition had no effect on Ha-Ras-induced stimulation of
R-Smad signalling, COL1A2 promoter transcription or collagen I accumulation (Figure
2D-F). Time-point analyses further revealed that R-Smad stimulation is transient and peaks
6 h after mDF infection, whereas collagen I protein levels remain elevated up to 24
h (Figure 3A and 3B). Interestingly, Ha-Ras-expressing cells also exhibited high protein levels of non-phosphorylated
Smad3 at 6 h and 24 h post-infection, which were not, however, associated with increased
levels of transcripts coding for Smad3 (Figure 3C). We interpreted these last results to suggest that greater amounts of available
Smad3 protein may also contribute to heightened pSmad3.

Figure 2.Activated phenotype of mouse dermal fibroblast (mDF) constitutively expressing Ha-Ras. (A) Left panel, pSmad3, Smad3, pERK1/2, ERK1/2, Ha-Ras and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) immunoblots of protein extracts from mDF cultures infected with
Ha-Ras (Ha-Ras lenti) or control (lenti) lentiviruses at 6 h post-infection (n = 3 per each sample) with the bar graphs on the side summarizing the relative ratios
of pSmad3 over GADPH and pERK1/2 over ERK1/2; right panel, luciferase activity of
a Smad3-responsive plasmid transiently transfected in mDF infected with Ha-Ras or
control lentivirus and/or stimulated with recombinant transforming growth factor-β
(TGFβ1; 2 ng/mL) for 6 h. (B) Collagen I and Ha-Ras immunoblots of protein extracts
from mDF cultures infected with Ha-Ras or control lentiviruses at 24 h post-infection
and in control cells treated with recombinant TGFβ1 (2 ng/mL) for the same length
of time (n = 3 per each sample). (C) α-smooth muscle actin (αSMA) immunostaining of mDF infected
with Ha-Ras or control lentiviruses with bar graphs on the side summarizing the percentage
of αSMA-positive cells 24 h after the infection; measurements were performed on >100
cells from three independent infections. (D) pSmad3 immunoblots of protein extracts
from mDF cultures treated with the MEK inhibitor PD98059 (10 μM) for 2 h prior to
infection for 6 h with Ha-Ras or control lentiviruses with bar graphs on the side
summarizing the relative ratio of pSmad3 and the loading control GADPH in the various
experimental samples (n = 3 per each sample). (E) Collagen I immunoblots of protein extracts from mDF cultures
treated with the MEK inhibitor PD98059 (10 μM) for 2 h prior to infection with Ha-Ras
or control lentiviruses (n = 3 per each sample); protein levels were assessed 24 h post-infection and the bar
graphs on the side summarize the relative ratio of collagen I and the loading control
GADPH in the various experimental samples. (F) Luciferase activity (expressed as fold
induction over control sample) of the COL1A2 promoter plasmid transiently transfected
in primary mDF infected with Ha-Ras or control lentiviruses at 24 h post-infection
with or without 2 h pre-treatment with the MEK inhibitor PD98059 (10 μM). In the relevant
panels, arrows and arrowheads point to Ha-Ras and a non-specific immunoreactive products
or pSmad3 and an unidentified cross-reacting R-Smad, respectively; luciferase values
represent the average of three independent transfections each performed in duplicate;
error bars signify ± standard deviation and asterisks indicate statistically significant
differences (P < 0.05).

Figure 3.pSmad3 and collagen type I kinetics in Ha-Ras-infected mouse dermal fibroblast (mDF). (A) pSmad3 and Smad3 and (B) collagen I immunoblots (n = 3 per each sample) of protein extracts prepared at the indicated time points after
infection of mDF cultures with Ha-Ras or control lentiviruses with bar graphs on the
side summarizing the ratios of pSmad3 or collagen I relative to the loading control
glyceraldehyde 3-phosphate dehydrogenase (GADPH) in the various experimental samples;
asterisks indicate statistically significant differences (P < 0.05). (C) Real-time quantitative polymerase chain reaction (qPCR) estimates of
Smad3 transcript levels in mDF cultures infected with Ha-Ras or control lentiviruses.
In the first panel, the arrow and arrowhead respectively point to pSmad3 and an unidentified
cross-reacting receptor-activated Smad (R-Smad).

Several reports have shown that Ha-Ras promotes autocrine TGFβ signalling [6,7,20]. The possible contribution of Ha-Ras to TGFβ production and, in turn, collagen I
synthesis was therefore investigated in our cell culture systems. To this end, a pan-TGFβ
antibody was employed in two complementary experiments which documented the inability
of TGFβ antagonism to normalize COL1A2 promoter activity in quiescent hDF cultures
transiently co-transfected with the ca-Ras expressing plasmid and in mDF cells stably
infected with the Ha-Ras lentivirus (Figure 4A and 4B). These findings excluded the potential involvement of TGFβ neo-synthesis in Ha-Ras
up-regulation of collagen I expression. Next, we evaluated whether Ha-Ras overexpression
may promote improper activation of latent TGFβ complexes as opposed to TGFβ neo-synthesis.
A bioassay, however, revealed comparable levels of active TGFβ in control and Ha-Ras
over-expressing mDF cells (Figure 5A). Consistent with this and the above findings, qPCR analyses showed similar levels
of TGFβ transcripts in control and Ha-Ras over-expressing mDF cells (Figure 5B). Collectively, the data suggested that increase of Ha-Ras protein levels in dermal
fibroblasts promotes an immediate fibrotic response through the canonical R-Smad pathway
without establishing an autocrine TGFβ loop.

Figure 4.Ha-Ras up-regulates collagen I independently of autocrine transforming growth factor-β
(TGFβ). (A) Luciferase activity (expressed as fold induction over control sample) of the
human pro-α2 (I) collagen gene (COL1A2) promoter co-transfected with the ca-Ras expressing
plasmid in quiescent human dermal fibroblast (hDF) cultured in the presence or absence
of TGFβ neutralizing antibody (2 and 10 μg/mL); cells stimulated with recombinant
TGFβ1 (2 ng/mL) served as a positive control. Luciferase values represent the average
of three independent transfections each performed in duplicate and error bars signify
± standard deviation and asterisks indicate statistically significant differences
(P < 0.05). (B) Collagen I immunoblots of protein extracts (n = 3 per each sample) from mouse dermal fibroblast (mDF) cultures infected with Ha-Ras
or control lentiviruses and cultured in the presence or absence of TGFβ neutralizing
antibody (10 μg/mL) for the indicated times. (C) pSmad3 immunoblots of protein extracts
(n = 3 per each sample) from mDF cultures stimulated with recombinant TGFβ1 (2 ng/mL)
in the presence or absence of neutralizing TGFβ antibodies (10 μg/mL). In the last
two panels, bar graphs summarize the ratio of pSmad3 or collagen I relative to the
loading control glyceraldehyde 3-phosphate dehydrogenase (GADPH) in the various experimental
samples; asterisks indicate statistically significant differences (P < 0.05).

Discussion

Accumulation of myofibroblasts and disorganized ECM are the hallmarks of tissue fibrosis.
TGFβ is a potent inducer of ECM synthesis and myofibroblasts contraction and a key
mediator of wound healing and fibrotic responses [2]. However, TGFβ pleiotropy has largely limited therapeutical intervention in fibrotic
diseases, thus stimulating an increased interest in the identification of pro-fibrotic
pathways that operate downstream, upstream or in parallel with TGFβ signalling [2]. Data presented here implicate Ha-Ras stabilization in the early onset of fibrosis
through TGFβ-independent stimulation of R-Smad signalling.

Previous reports that Ha-Ras proto-oncogene intersects with TGFβ signalling events
in several fibrotic conditions [8,9], together with recent evidence that scleroderma auto-antibodies stabilize Ha-Ras
levels through ROS action [15,16], led us to hypothesize that increased Ha-Ras activity may influence TGFβ signalling
during the early phase of the pro-fibrotic response. Through Ha-Ras overexpression
in quiescent mDF, we have demonstrated that the proto-oncogene directly up-regulates
collagen production through TGFβ-independent activation of the canonical R-Smad pathway.

Three independent lines of evidence support our conclusion. First, forced expression
of DN-Smad3 and mutations in the TbRE of the COL1A2 promoter significantly reduced
Ha-Ras-dependent up-regulation of the reporter plasmid. Second, lentiviral overexpression
of Ha-Ras rapidly increased Smad3 signalling and R-Smad-dependent reporter activity.
Third, mDF pre-treatment with neutralizing pan-TGFβ antibody, or with a MEK inhibitor,
showed no appreciable effects on collagen accumulation and Ha-Ras-induced R-Smad activation.
These results are in agreement with previous reports indicating that ligand-independent
R-Smad signalling is increased in scleroderma cells and that Ha-Ras-dependent MAPK
stimulation is not required for R-Smad activation [10,21]. In contrast to the reported participation of ERK1/2 signalling in perpetuating ROS
and Ha-Ras stimulation of collagen I production in SSc fibroblasts [15,16], ERK1/2 signalling is not required for collagen I up-regulation in Ha-Ras over-expressing
cells. This apparent discrepancy suggests that constitutively high levels of Ha-Ras
in our cell culture system do not require the postulated feed-forward loop of ERK1/2
signalling [15,16]; implicitly, our conclusion suggests that the amount of Ha-Ras is a limiting factor
during the early phase of SSc fibrosis.

Protein turnover is another modulator of R-Smad3 activity in addition to protein phosphorylation
[4]. The finding that Ha-Ras overexpression in mDF is associated with increased Smad3
protein levels and normal amounts of Smad3 transcripts strongly suggests decreased protein degradation. Recently, the protein
kinase GSK3β has been shown to control Smad3 ubiquitination and degradation [22]. GSK3β activity is negatively regulated by Ras family members and fibroblast-specific
deletion of GSK3β in mice results in accelerated wound closure, increased fibrogenesis
and excessive scarring [23,24]. Altogether, these reports and our results are at least consistent with the notion
that augmented Smad stability in mDF overexpressing Ha-Ras accounts in part for the
ligand-independent increase of canonical TGFβ signalling and collagen production.
A similar situation has been described for SSc fibroblasts in which higher than normal
levels of Smad3 and Ha-Ras proteins are both associated with increased collagen accumulation
[16,21].

In contrast with our observations, others have reported that oncogenic Ha-Ras (V12-Ha-Ras) dictates the response of cancer cells to TGFβ by decreasing Smad3 stability,
thus suggesting a negative role of Ha-Ras in modulating R-Smad signalling [25]. Moreover, Ha-Ras has been shown to inhibit R-Smad activity in epithelial cells and
to down-regulate collagen expression in proliferating fibroblasts [26,27]. We believe that these apparent discrepancies probably reflect the multiple roles
that Ha-Ras plays in integrating the contextual responses of cells to TGFβ signalling.
Our results contribute to the ongoing efforts to dissect the complex network of signalling
events that drive the onset and progression of tissue fibrosis.

Conclusions

Our results provide a mechanistic insight into the role of Ha-Ras stabilization in
driving collagen I overproduction during the early phase of dermal fibrosis in SSc
by showing a direct involvement of the proto-oncogene in stimulating R-Smad signalling
independently of TGFβ neo-synthesis or activation and of ERK1/2 signalling. This conclusion
is based on in vitro evidence that correlated Ha-Ras-induced activation of R-Smad directly to the stimulation
of COL1A2 promoter reporter plasmid and with the elevation of endogenous collagen
I protein. Together, our findings extend and refine recent reports that implicated
circulating PDFR auto-antibody in the triggering of the ROS-mediated stabilization
of Ha-Ras activity in SSc [15,16]. As such, they contribute to a better understanding of the early signalling events
and potential therapeutic opportunities in this acquired disabling disorder of the
connective tissue.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SS and JO carried out all the experiments and data analyses. LG provided the lentiviral
vectors and relevant protocols, in addition to supervising the lentivirus infection
experiments. GM performed TGFβ assays with scleroderma auto-antibodies that were not
included in the present manuscript. AG and FR designed the experiments and interpreted
the data. SS and FR wrote the final manuscript. All authors have read and approved
the final manuscript.

Acknowledgements

We are grateful to Dr M Kypriotou for helping with some early experiments and to Ms
K Johnson for organizing the manuscript. The work was supported from grants of the
National Institutes of help (AR055806) and the Scleroderma Foundation.